Tài liệu Báo cáo Y học: Steady-state kinetics of the glutaminase reaction of CTP synthase from

Steady-state kinetics of the glutaminase reaction of CTP synthase

from Lactococcus lactis

The role of the allosteric activator GTP in coupling between glutamine hydrolysis

and CTP synthesis

Martin Willemoe¨ s1 and Bent W. Sigurskjold2

1

Centre for Crystallographic Studies, Department of Chemistry, University of Copenhagen, Copenhagen, Denmark;

2

Department of Biochemistry, August Krogh Institute, University of Copenhagen, Copenhagen, Denmark

CTP synthase catalyzes the reaction glutamine + UTP

+ ATP fi glutamate + CTP + ADP + Pi

. The rate

of the reaction is greatly enhanced by the allosteric activator

GTP. We have studied the glutaminase half-reaction of CTP

synthase from Lactococcus lactis and its response to the

allosteric activator GTP and nucleotides that bind to

the active site. In contrast to what has been found for the

Escherichia coli enzyme, GTP activation of the L. lactis

enzyme did not result in similar kcat values for the glutami￾nase activity and glutamine hydrolysis coupled to CTP

synthesis. GTP activation of the glutaminase reaction never

reached the levels of GTP-activated CTP synthesis, not even

when the active site was saturated with UTP and the non￾hydrolyzeable ATP-binding analog adenosine 5¢-[c-thio]tri￾phosphate. Furthermore, under conditions where the rate of

glutamine hydrolysis exceeded that of CTP synthesis, GTP

would stimulate CTP synthesis. These results indicate that

the L. lactis enzyme differs significantly from the E . coli

enzyme. For the E . coli enzyme, activation by GTP was

found to stimulate glutamine hydrolysis and CTP synthesis

to the same extent, suggesting that the major function of

GTP binding is to activate the chemical steps of glutamine

hydrolysis. An alternative mechanism for the action of GTP

on L. lactis CTP synthase is suggested. Here the binding of

GTP to the allosteric site promotes coordination of the

phosphorylation of UTP and hydrolysis of glutamine for

optimal efficiency in CTP synthesis rather than just acting to

increase the rate of glutamine hydrolysis itself.

Keywords: CTP synthase; isothermal titration calorimetry;

glutaminase activity; allosteric regulation;Lactococcus lactis.

CTP synthase (EC 6.4.3.2) catalyzes the synthesis of CTP

from UTP by amination of the pyrimidine ring at the

4-position. The enzyme has three functionally distinct sites;

the glutaminase site where glutamine hydrolysis occurs, the

active site where CTP synthesis takes place and the allosteric

site that binds GTP. The reaction is thought to proceed via

phosphorylation of UTP by ATP to give an activated

intermediate 4-phosphoryl UTP and ADP [1,2]. Ammonia

then reacts with this intermediate yielding CTP and Pi as

illustrated in Scheme 1A. Ammonia can either be utilized

directly or be generated from the hydrolysis of glutamine in

a GTP-activated reaction [3,4]. Similar mechanisms to that

shown in Scheme 1A, have been shown now for several

amido transferase enzymes [5–7]. Here the binding of an

already activated substrate, or activation of the substrate on

the enzyme, in this case by phosphorylation, precedes

amination. The overall reaction is as follows:

*

PPPrib

O

HN

N

OPO3

2-

PPPrib

O

O

HN

N

ATP NH3

PPPrib

H2N

O

HN

N

OPO3

2-

PPPrib

NH2

O

N

N

A

ADP Pi

PPPrib

O

O

HN

N

PPPrib

H2N

O

HN

N

OPO3

2-

PPPrib

NH2

O

N

N

B

NH3* ATP

ADP Pi

PPPrib

H2N

O

HN

N

OH

Scheme 1. Proposed mechanisms of CTP synthesis. The box indicates

an expected transition state like structure. * indicates that the amino

donor can either be free ammonia or ammonia generated from

hydrolysis of glutamine.

Correspondence to M. Willemoe¨s, Centre for Crystallographic Studies,

Department of Chemistry, University of Copenhagen,

Universitetsparken 5, DK-2100 Copenhagen Ø, Denmark.

Fax: + 45 35320299, Tel.: + 45 35320239,

E-mail: [email protected]

Abbreviations: ADPNP, adenosine 5¢-[b,c-imido]triphosphate;

ATP-cS, adenosine 5¢-[c-thio]triphosphate; CPS, carbamoyl

phosphate synthase; DON, 6-diazo-5-oxo-norleucine; GDH,

glutamate dehydrogenase; ITC, isothermal titration calorimetry.

Enzymes: CTP synthase (EC 6.4.3.2).

(Received 24 May 2002, revised 29 July 2002, accepted 9 August 2002)

Eur. J. Biochem. 269, 4772–4779 (2002) FEBS 2002 doi:10.1046/j.1432-1033.2002.03175.x